Avidin is a homotetrameric glycoprotein isolated from chicken egg-white. Each of the eight-stranded beta-barrel subunit of avidin consists of 128 amino acids and has one ligand-binding site. Avidin, like its bacterial analogue streptavidin from Streptomyces avidinii, is able to form a tight and specific complex with a water-soluble vitamin, d-biotin (dissociation constant Kd≈10−15 M) (1,2). This special property of avidin together with its tetrameric nature and high stability have made it one of the most widely exploited protein tools in the life-sciences across a range of biochemical, pharmaceutical and biophysical applications (3,4).
The avidin gene family consists of avidin and seven avidin-related genes (AVRs) (5). Although avidin protein is expressed in various tissues (6), the other members of the gene family have not so far been found in the form of proteins in the chicken. In order to study their functional and structural properties, AVR proteins were recently produced by a baculovirus insect cell expression system (7). Genes AVR4 and AVR5 both encode identical protein, called AVR4/5. Avidin and AVR4/5 are about 80% identical in amino acid sequence, and almost all of the residues involved in biotin binding in avidin are conserved in AVR4/5. It was found that recombinant AVR4/5 bound biotin almost as tightly as avidin. Most interestingly, it was shown to be significantly more thermostable, the transition midpoint of heat denaturation Tm being 106.4° C. compared to that of avidin, which Tm is 83.5° C. (8).
It has been proposed that protein oligomerization in nature serves to obtain more stable structures (9,10). In addition, stable proteins tend to have only a few intrinsic water clefts in their structures (11,12). Moreover, the role of ionic bonds in establishing the high thermal stability of proteins has been studied by Szilágyi and Závodszky (13), who performed a statistical analysis of high-quality protein structures obtained from mesophilic and thermophilic organisms. They observed a correlation between the number of ion pairs and growth temperature of the organism, and hypothesized that ion pairs have structural importance especially at high temperatures. The ionic bonds found in thermostable proteins have successfully been transferred to their analogues from mesophilic organisms in order to stabilize them (14). The importance of aromatic pairs in thermostable proteins has also been noticed (15), and these pairs have successfully been transferred between proteins to improve the thermal stability (16).